[Research Progress of the Correlation between Caveolin and Unexpected Sudden Cardiac Death].

Due to the negative autopsy and without cardiac structural abnormalities, unexpected sudden cardiac death （USCD） is always a tough issue for forensic pathological expertise. USCD may be associated with parts of fatal arrhythmic diseases. These arrhythmic diseases may be caused by disorders of cardiac ion channels or channel-related proteins. Caveolin can combine with multiple myocardial ion channel proteins through its scaffolding regions and plays an important role in maintaining the depolarization and repolarization of cardiac action potential. When the structure and function of caveolin are affected by gene mutations or abnormal protein expression, the functions of the regulated ion channels are correspondingly impaired, which leads to the occurrence of multiple channelopathies, arrhythmia or even sudden cardiac death. It is important to study the effects of caveolin on the functions of ion channels for exploring the mechanisms of malignant arrhythmia and sudden cardiac death.

[Correlation between Genetic Variants and Polymorphism of Caveolin and Sudden Unexplained Death].

OBJECTIVES: To explore the genetic variation sites of caveolin （CAV） and their correlation with sudden unexplained death （SUD）. METHODS: The blood samples were collected from SUD group （71 cases）, coronary artery disease （CAD） group （62 cases） and control group （60 cases）, respectively. The genome DNA were extracted and sequencing was performed directly by amplifying gene coding region and exon-intron splicing region of CAV1 and CAV3 using PCR. The type of heritable variation of CVA was confirmed and statistical analysis was performed. RESULTS: A total of 4 variation sites that maybe significative were identified in SUD group, and two were newfound which were CAV1： c.45C>T （T15T） and CAV1：c.512G>A （R171H）, and two were SNP loci which were CAV1：c.246C>T （rs35242077） and CAV3：c.99C>T （rs1008642） and had significant difference （P<0.05） in allele and genotype frequencies between SUD and control groups. Forementioned variation sites were not found in CAD group. CONCLUSIONS: The variants of CAV1 and CAV3 may be correlated with a part of SUD group.

Cross-clade HIV-1 neutralizing antibodies induced with V3-scaffold protein immunogens following priming with gp120 DNA.

The V3 epitope is a known target for HIV-1 neutralizing antibodies (NAbs), and V3-scaffold fusion proteins used as boosting immunogens after gp120 DNA priming were previously shown to induce NAbs in rabbits. Here, we evaluated whether the breadth and potency of the NAb response could be improved when boosted with rationally designed V3-scaffold immunogens. Rabbits were primed with codon-optimized clade C gp120 DNA and boosted with one of five V3-cholera toxin B fusion proteins (V3-CTBs) or with double combinations of these. The inserts in these immunogens were designed to display V3 epitopes shared by the majority of global HIV-1 isolates. Double combinations of V3-CTB immunogens generally induced more broad and potent NAbs than did boosts with single V3-CTB immunogens, with the most potent and broad NAbs elicited with the V3-CTB carrying the consensus V3 of clade C (V3(C)-CTB), or with double combinations of V3-CTB immunogens that included V3(C)-CTB. Neutralization of tier 1 and 2 pseudoviruses from clades AG, B, and C and of peripheral blood mononuclear cell (PBMC)-grown primary viruses from clades A, AG, and B was achieved, demonstrating that priming with gp120 DNA followed by boosts with V3-scaffold immunogens effectively elicits cross-clade NAbs. Focusing on the V3 region is a first step in designing a vaccine targeting protective epitopes, a strategy with potential advantages over the use of Env, a molecule that evolved to protect the virus by poorly inducing NAbs and by shielding the epitopes that are most critical for infectivity.

Mutational analysis and an alternatively spliced product of B7 defines its CD28/CTLA4-binding site on immunoglobulin C-like domain.

Costimulatory molecules B7 and B7-2 interact with T cell surface receptors CD28/CTLA4 and deliver a costimulatory signal essential for T cell growth. However, the structure basis of this interaction is not known. B7 and B7-2 are members of immunoglobulin (Ig) superfamily and their extracellular portion consists of an IgV- and IgC-like domain. Here we report that a naturally occurring, alternatively spliced form of B7 reveals that exon 3-encoded IgC domain is essential for CD28/CTLA4 binding. Mutational analysis of B7 demonstrates a critical role of several amino acids around loops between strands B and C and D and E, for binding CTLA4/CD28. These amino acids are clustered to form a single binding site centered at 201Y. A comparison of the effects of mutations on the binding of CD28 and CTLA4 reveals that CD28 and CTLA4 binds to the same site on B7. These results have important implications on the role of CTLA4 and CD28 in T cell costimulation. The structure of the CD28/CTLA4-binding site also provides valuable information for immune intervention targeted at the B7/B7-2-CD28/CTLA4 interactions.

Enhanced susceptibility of Cpt1c knockout mice to glucose intolerance induced by a high-fat diet involves elevated hepatic gluconeogenesis and decreased skeletal muscle glucose uptake.

AIMS/HYPOTHESIS: Carnitine palmitoyltransferase-1 (CPT1)c is a novel isoform in the CPT1 family and is found specifically in the brain. Cpt1c knockout (KO) mice are more susceptible to high-fat diet (HFD)-induced obesity. However, the underlying mechanism of this phenotype and the question of whether CPT1c is involved in the pathogenesis of diet-induced insulin resistance are unclear. METHODS: To assess the potential role of CPT1c in the regulation of whole-body glucose homeostasis, we generated Cpt1c KO mice and challenged them with HFD or standard chow. Glucose homeostasis of each group was assessed weekly. RESULTS: After 8 weeks of HFD feeding, Cpt1c KO mice developed a phenotype of more severe insulin resistance than that in wild-type controls. The increased susceptibility of Cpt1c KO mice to HFD-induced insulin resistance was independent of obesity. Impaired glucose tolerance in Cpt1c KO mice was attributable to elevated hepatic gluconeogenesis and decreased glucose uptake in skeletal muscle. These effects correlated with decreased hepatic and intramuscular fatty acid oxidation and expression of oxidative genes as well as with elevated triacylglycerol content in these tissues. Interestingly, Cpt1c deletion caused a specific elevation of hypothalamic CPT1a and CPT1b isoform expression and activity. We demonstrated that elevated plasma NEFA concentration is one mechanism via which this compensatory effect is induced. CONCLUSIONS/INTERPRETATION: These results further establish the role of CPT1c in controlling whole-body glucose homeostasis and in the regulation of hypothalamic Cpt1 isoform expression. We identify changes in hepatic and skeletal muscle glucose metabolism as important mechanisms determining the phenotype of Cpt1c KO mice.

Crystallization of proliferating cell nuclear antigen (PCNA) from Saccharomyces cerevisiae.

Proliferating cell nuclear antigen (PCNA) is the component of the chromosomal DNA replication machinery in eukaryotic cells that confers high processivity upon DNA polymerase delta and epsilon. It has been proposed that PCNA functions by forming a trimeric complex with a ring-like structure through which DNA is threaded. PCNA from the yeast Saccharomyces cerevisiae has been crystallized in a cubic space group (P2(1)3, a = 121.1 A). Unexpectedly, a mercury derivative of PCNA yields crystals that diffract significantly better than crystals of the unmodified protein (2.4 A and 3.0 A resolution, respectively). Mass spectrometry reveals that the derivative results from the addition of two mercury atoms to the protein. Although crystals of the mercurated protein show evidence of non-isomorphism, the anomalous diffraction signal is strong and phases may be determined by multi-wavelength anomalous diffraction (MAD phasing).

A novel, picomolar inhibitor of human immunodeficiency virus type 1 protease.

The design, synthesis, and molecular modeling studies of a novel series of azacyclic ureas, which are inhibitors of human immunodeficiency virus type 1 (HIV-1) protease that incorporate different ligands for the S1', S2, and S2' substrate-binding sites of HIV-1 protease are described. The synthesis of this series is highly flexible in the sense that the P1', P2, and P2' residues of the inhibitors can be changed independently. Molecular modeling studies on the phenyl ring of the P2 and P2' ligand suggested incorporation of hydrogen-bonding donor/acceptor groups at the 3' and 4-positions of the phenyl ring should increase binding potency. This led to the discovery of compound 7f (A-98881), which possesses high potency in the HIV-1 protease inhibition assay and the in vitro MT-4 cell culture assay (Ki = approximately 5 pM and EC50 = 0.002 microM). This compares well with the symmetrical cyclic urea 1 pioneered at DuPont Merck.

[Preventive effect of cardiomyopeptidin on rat heart injured by ischemia-reperfusion].

OBJECTIVE: To investigate the preventive effect of cardiomyopeptidin of small molecular weight polypeptide on rat hearts injured by ischemia-reperfusion. METHODS: In a rat model injured by ischemia-reperfusion in the heart, observation was made on the influence of cardiomyopeptidin on the activities of creatine kinase (CK) and lactate dehydrogenase(LDH), as well as on the content of MDA in plasma after the preventive drug was used. RESULTS: Cardiomyopeptidin could obviously prevent the injury caused by ischemia-reperfusion, reduce the activities of CK and LDH and the content of MDA in a dose-dependent manner. CONCLUSION: Cardiomyopeptidin has a preventive effect on myocardium injured by ischemia-reperfusion and this may be related to its reducing the release of myocardial enzyme and anti-lipoperoxidation.

[Recovery effect of cardiomyopeptidin fractions and fraction addition on cardiac muscle cells in rats damaged by adriamycin].

OBJECTIVE: To investigate the recovery effect of cardiomyopeptidin fractions and fraction addition on the cardiac muscle cells in rats damaged by adriamycin. METHODS: Observing the activity of the succinic dehydrogenase which is at mitochondrion in the cells damaged by adriamycin with MTT. RESULTS: Five fractions have all promoted the activating effect of the enzyme, the action of PI being higher than the others. Fraction addition has also promoted the activating effect of the enzyme, but without additive effect. CONCLUSION: The recovery effect of cardimyopeptidin depends on the interplay among the fractions.

Neuropeptide S promotes wakefulness through activation of the posterior hypothalamic histaminergic and orexinergic neurons.

In spite of the initial and pivotal findings that the newly identified neuropeptide S (NPS) promotes arousal associated with locomotor and anxiolytic-like effects, the mechanisms through which NPS acts to modulate sleep-waking states remain unclear. The present study was undertaken to investigate in the rat the effects of i.c.v. injection of NPS on the EEG, sleep-wake cycle, and brain c-Fos expression. NPS at 0.1 and 1 nmol increased significantly wakefulness (W) during the first 2 h (54.7 ± 3.2 and 64.9 ± 2.1 min, respectively, vs. 41.4 ± 2.5 min seen with saline injections, P<0.01 and P<0.001), accompanied by an increase in EEG high frequency activities (14.5-60 Hz). In the meanwhile, slow wave sleep (SWS) and paradoxical sleep (PS) decreased significantly. Ex-vivo Fos immunohistochemistry in the posterior hypothalamus revealed that, as compared with saline-treated rats, NPS enhanced c-Fos expression in histaminergic neurons by 76.0% in the ventral tuberomammillary nucleus (TMN) and 57.8% in the dorsal TMN, and in orexinergic neurons by 28.2% in the perifornical nucleus (PeF), 24.3% in the dorsomedial hypothalamic nucleus (DMH), and 13.7% in the lateral hypothalamic area (LH) of the posterior hypothalamus. The NPS-induced c-Fos expression in histaminergic neurons and orexinergic neurons where NPS receptor (NPSR) mRNA is highly expressed, suggests that NPS activates histaminergic and orexinergic neurons to promote W.

Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA.

The crystal structure of the processivity factor required by eukaryotic DNA polymerase delta, proliferating cell nuclear antigen (PCNA) from S. cerevisiae, has been determined at 2.3 A resolution. Three PCNA molecules, each containing two topologically identical domains, are tightly associated to form a closed ring. The dimensions and electrostatic properties of the ring suggest that PCNA encircles duplex DNA, providing a DNA-bound platform for the attachment of the polymerase. The trimeric PCNA ring is strikingly similar to the dimeric ring formed by the beta subunit (processivity factor) of E. coli DNA polymerase III holoenzyme, with which it shares no significant sequence identity. This structural correspondence further substantiates the mechanistic connection between eukaryotic and prokaryotic DNA replication that has been suggested on biochemical grounds.

Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp.

The crystal structure of the beta subunit (processivity factor) of DNA polymerase III holoenzyme has been determined at 2.5 A resolution. A dimer of the beta subunit (M(r) = 2 x 40.6 kd, 2 x 366 amino acid residues) forms a ring-shaped structure lined by 12 alpha helices that can encircle duplex DNA. The structure is highly symmetrical, with each monomer containing three domains of identical topology. The charge distribution and orientation of the helices indicate that the molecule functions by forming a tight clamp that can slide on DNA, as shown biochemically. A potential structural relationship is suggested between the beta subunit and proliferating cell nuclear antigen (PCNA, the eukaryotic polymerase delta [and epsilon] processivity factor), and the gene 45 protein of the bacteriophage T4 DNA polymerase.

Crystal structure of human stem cell factor: implication for stem cell factor receptor dimerization and activation.

Stem cell factor (SCF) plays important roles in hematopoiesis and the survival, proliferation, and differentiation of mast cells, melanocytes, and germ cells. SCF mediates its biological effects by binding to and activating a receptor tyrosine kinase designated c-kit or SCF receptor. In this report we describe the 2.3-A crystal structure of the functional core of recombinant human SCF. SCF is a noncovalent homodimer composed of two slightly wedged protomers. Each SCF protomer exhibits an antiparallel four-helix bundle fold. Dimerization is mediated by extensive polar and nonpolar interactions between the two protomers with a large buried surface area. Finally, we have identified a hydrophobic crevice and a charged region at the tail of each protomer that functions as a potential receptor-binding site. On the basis of these observations, a model for SCF small middle dotc-kit complex formation and dimerization is proposed.

X-ray structure of trypanothione reductase from Crithidia fasciculata at 2.4-A resolution.

Trypanosomes and related protozoan parasites lack glutathione reductase and possess instead a closely related enzyme that serves as the reductant of a bis(glutathione)-spermidine conjugate, trypanothione. The human and parasite enzymes have mutually exclusive substrate specificities, providing a route for the design of therapeutic agents by specific inhibition of the parasite enzyme. We report here the three-dimensional structure of trypanothione reductase from Crithidia fasciculata and show that it closely resembles the structure of human glutathione reductase. In particular, the core structure surrounding the catalytic machinery is almost identical in the two enzymes. However, significant differences are found at the substrate binding sites. A cluster of basic residues in glutathione reductase is replaced by neutral, hydrophobic, or acidic residues in trypanothione reductase, consistent with the nature of the spermidine linkage and the change in overall charge of the substrate from -2 to +1, respectively. The binding site is more open in trypanothione reductase due to rotations of about 4 degrees in the domains that form the site, with relative shifts of as much as 2-3 A in residue positions. These results provide a detailed view of the residues that can interact with potential inhibitors and complement previous modeling and mutagenesis studies on the two enzymes.

Uroplakin Ia is the urothelial receptor for uropathogenic Escherichia coli: evidence from in vitro FimH binding.

The binding of uropathogenic Escherichia coli to the urothelial surface is a crucial initial event for establishing urinary tract infection because it allows the bacteria to gain a foothold on the urothelial surface, thus preventing them from being removed by micturition. In addition, it triggers bacterial invasion as well as host urothelial defense. This binding is mediated by the FimH adhesin located at the tip of the bacterial type 1-fimbrium, a filamentous attachment apparatus, and its urothelial receptor. We have prepared a biotinylated, recombinant FimH-FimC adhesin:chaperone complex and used it to identify its mouse urothelial receptor. The FimH-FimC complex binds specifically to a single 24 kDa major mouse urothelial plaque protein, which we identified as uroplakin Ia by mass spectrometry, cDNA cloning and immunoreactivity. The terminal mannosyl moieties on Asn-169 of uroplakin Ia are responsible for FimH as well as concanavalin A binding. Although FimH binds to uroplakin Ia with only moderate strength (K(d) approximately 100 nM between pH 4 and 9), the binding between multiple fimbriae of a bacterium and the crystalline array of polymerized uroplakin receptors should achieve high avidity and stable bacterial attachment. The FimH-FimC complex binds preferentially to the mouse urothelial umbrella cells in a pattern similar to uroplakin staining. Our results indicate that the structurally related uroplakins Ia and Ib are glycosylated differently, that uroplakin Ia serves as the urothelial receptor for the type 1-fimbriated E. coli, and that the binding of uropathogenic bacteria to uroplakin Ia may play a key role in mediating the urothelial responses to bacterial attachment.

Organization of uroplakin subunits: transmembrane topology, pair formation and plaque composition.

The apical surfaces of urothelial cells are almost entirely covered with plaques consisting of crystalline, hexagonal arrays of 16 nm uroplakin particles. Although all four uroplakins, when SDS-denatured, can be digested by chymotrypsin, most uroplakin domains in native urothelial plaques are resistant to the enzyme, suggesting a tightly packed structure. The only exception is the C-terminal, cytoplasmic tail of UPIII (UPIII) which is highly susceptible to proteolysis, suggesting a loose configuration. When uroplakins are solubilized with 2% octylglucoside and fractionated with ion exchangers, UPIa and UPII were bound as a complex by a cation exchanger, whereas UPIb and UPIII were bound by an anion exchanger. This result is consistent with the fact that UPIa and UPIb are cross-linked to UPII and UPIII, respectively, and suggests that the four uroplakins form two pairs consisting of UPIa/II and UPIb/III. Immunogold labelling using a new mouse monoclonal antibody, AU1, revealed that UPIII is present in all urothelial plaques, indicating that the two uroplakin pairs are not segregated into two different types of urothelial plaque and that all plaques must have a similar uroplakin composition. Taken together, these results indicate that uroplakins form a tightly packed structure, that the four uroplakins interact specifically forming two pairs, and that both uroplakin pairs are required for normal urothelial plaque formation.

ABT-538 is a potent inhibitor of human immunodeficiency virus protease and has high oral bioavailability in humans.

Examination of the structural basis for antiviral activity, oral pharmacokinetics, and hepatic metabolism among a series of symmetry-based inhibitors of the human immunodeficiency virus (HIV) protease led to the discovery of ABT-538, a promising experimental drug for the therapeutic intervention in acquired immunodeficiency syndrome (AIDS). ABT-538 exhibited potent in vitro activity against laboratory and clinical strains of HIV-1 [50% effective concentration (EC50) = 0.022-0.13 microM] and HIV-2 (EC50 = 0.16 microM). Following a single 10-mg/kg oral dose, plasma concentrations in rat, dog, and monkey exceeded the in vitro antiviral EC50 for > 12 h. In human trials, a single 400-mg dose of ABT-538 displayed a prolonged absorption profile and achieved a peak plasma concentration in excess of 5 micrograms/ml. These findings demonstrate that high oral bioavailability can be achieved in humans with peptidomimetic inhibitors of HIV protease.